This invention relates to pharmaceutical compositions and methods of using non-peptidic cyclophilin-binding compounds in medical conditions involving breakdown of mitochondrial energy metabolism induced by calcium overload, in treating alopecia and promoting hair growth, in treating infections with filarial and helmintic parasites, and in treating and preventing infections with the human immunodeficiency virus.
The cyclic undecapeptide cyclosporin A (CyA), as well as two other drugs, FK-506 and rapamycin, are well-known in the art as potent T-cell specific immunosuppressants, and are effective against graft rejection after organ transplantation. In vivo and in vitro, these compounds bind to two distinct classes of proteins commonly known as immunophilins. Cyclophilins (CyP), which bind cyclosporin A, and FK506-binding proteins (FKBP), which bind FK-506 and rapamycin, are both subclasses of this group of proteins termed immunophilins. Immunophilins were first identified as proteins that bind to the immunosuppressive drugs cyclosporin A, FK506, and rapamycin. CyPs and FKBPs can also be separated based on their differing structures.
The effects of the cyclosporin A:cyclophilin interaction have been well documented. Cyclosporin A binds with a dissociation constant in the range of 10xe2x88x928 mol/L, a value representing a relatively high degree of attraction (Handschumacher et al., Science 226:544 (1984)). While the present invention is not bound by any particular theory, it appears the complex formed between CyP and cyclosporin A exerts the effects on the organism and cells, which leads to immunosuppression. The complex interacts with the cellular enzyme calcineurin, a calmodulin-dependent phosphatase, and the interaction prevents T cell activation by blocking RNA transcription of the T cell growth factor interleukin 2 (IL-2). (Palacios, J. Immunol. 128:337 (1982)). Without IL-2 to cause T cell proliferation, specific T cell populations cannot mount a strong immune response, resulting in immunosuppression.
A number of types of mammalian cyclophilins have been identified and cloned, cyclophilins A, B, C, D, and cyclophilin-40 (Snyder and Sabatini, Nat. Med. 1:32-37 (1995); Friedman et al., Proc. Natl. Acad. Sci., 90:6815-6819 (1993)). Cyclophilin A is a 19 kD protein, which is abundantly expressed in a wide variety of cells. Like the other cyclophilins, cyclophilin A binds the immunosuppressive agent cyclosporin A and possesses peptidyl-prolyl cis-trans isomerase (PPIase) and protein folding or xe2x80x9cchaperonexe2x80x9d activities. PPIase activity catalyzes the conversion of proline residues in a protein from the cis to the trans conformation (Fischer, et al., Biomed. Biochem. Acta 43:1101-1112 (1984)). Cyclophilin B possesses an N-terminal signal sequence that directs translocation into the endoplasmic reticulum of the cell. The 23 kD cyclophilin C is found in the cytosol of the cell. Cyclophilin D, at 18 kD, appears to target its actions in the mitochondria. And cyclophilin-40 is a component of the inactivated form of a glucocorticoid receptor.
Since immunophilins, including the cyclophilin group of proteins, were discovered because of their interaction with known immunosuppressive drugs, drug discovery efforts initially focused on improving the immunosuppressant potency, and optimizing the pharmacological profile, of cyclosporin A and its peptidic analogues for immunosuppressant uses. Later, other biological effects of immunosuppressant cyclophilin-binding drugs were discovered. It has been reported that, in murine models which mimic human premature hair follicle regression or human chemotherapy-induced hair loss, topical application of CsA induces and maintains hair growth, and topical or systemic administration of CsA protects from hair loss induced by cancer chemotherapeutic agents (see, e.g., Maurer, et al. Am. J. Pathol. 150(4): 1433-41 (1997); Paus, et al., Am. J. Pathol. 144, 719-34 (1994)). One form of hair loss, alopecia areata, is known to be associated with autoimmune biological processes; hence, topically administered immunomodulatory compounds are expected to be efficacious in treating this particular form of hair loss. However, there is evidence that initiation of hair growth by CsA is unrelated to immunosuppression (Iwabuchi, et al., J. Dermatol. Sci. 9, 64-69 (1995)).
FK506 has also been shown to stimulate hair growth in a dose-dependent manner when administered topically (Yamamoto, et al., J. Invest. Dermatol. 102 (1994) 160-164; Jiang, et al., J. Invest. Dermatol., 104 (1995) 523-525).
The use of cyclosporin A and related compounds for hair revitalization has been disclosed in U.S. Pat. No. 5,342,625 (Hauer et al.), U.S. Pat. No. 5,284,826 (Eberle), U.S. Pat. No. 4,996,193 (Hewitt et al.). These patents relate to compounds and compositions useful for treating immune-related disorders and cite the known use of cyclosporin and related immunosuppressive compounds for hair growth. The known utility of cyclosporin A in promoting hair growth has also been cited in earlier work by the present inventors, see, e.g., U.S. Pat. No. 6,172,087 B1 (Steiner and Hamilton), U.S. Pat. No. 6,177,455 B1 (Steiner and Hamilton), U.S. Pat. No. 6,187,784 B1 (Steiner and Hamilton).
Another biological activity of the cyclophilin-binding compounds cyclosporin and its peptidic analogues relates to their protective effects on proapoptotic cells. The mitochondrion is increasingly being recognized as an important mediator of cell death in hypoxia, ischemia, and chemical toxicity. Disruption of the mitochondrial transmembrane potential is observed before other features of apoptosis (e.g. generation of reactive oxygen species or internucleosomal DNA fragmentation (xe2x80x9cladderingxe2x80x9d)) become detectable. This applies to many different models of apoptosis induction, such as, for example, NGF-deprivation of cultured sympathetic neurons, dexamethasone-induced lymphocyte apoptosis, programmed lymphocyte death, activation-induced programmed cell death of T cell hybridomas, and tumor necrosis factor-induced death of lymphoma cells. [Marchetti, P., et al., J. Exp. Med. 184, 1996, 1155-1160]. Breakdown of mitochondrial transmembrane potential in proapoptotic cells has been attributed to the formation of an unspecific high conductance channelxe2x80x94the mitochondrial permeability transition porexe2x80x94which leads to an increased permeability of the inner mitochondrial membrane to small molecular weight solutes. The ensuing release of intramitochondrial ions, influx of solutes, uncoupling of oxidative phosphorylation, and loss of metabolic intermediates accompanies large amplitude mitochondrial swelling and a depletion of cellular energy stores [see, e.g., Lemasters, J. J. et al., Mol. Cell. Biochem. 174 (1997) 159-165]. Importantly, CsA and non-immunosuppressive peptidic CsA analogues have been described to potently block pore conductance and inhibit the onset of the mitochondrial permeability transition [Broekemeier, K. M., et al., J. Biol. Chem. 264 (1989) 7826-7830; Zamzami, M., et al., FEBS Lett. 384 (1996) 53-7]. The mitochondrial permeability transition pore forms under calcium overload conditions such as occur in ischemia/reperfusion injury, and it has been found that administration of CsA and/or non-immunosuppressive peptidic CsA analogues, by blocking the permeability transition pore, leads to significant protection in experimental models of cerebral stroke [Matsumoto, S., et al., J. Cereb. Blood Flow Metab. 19 (1999) 736-41], cardiac ischemia [Griffiths, E. J. and Halestrap, A. P., J. Mol. Cell Cardiol. 25 (1993) 1461-1469], and hepatic ischemia/reperfusion injury [Leducq, N., et al., Biochem. J. 336 (1998) 501-6 ].
CsA and its non-immunosuppressive peptidic analogues have also been found to potently inhibit the growth of pathogenic protozoan parasites, such as Cryptosporidium parvum, Plasmodium falciparum, Plasmodium vivax, Schistosoma spec., and Toxoplasma gondii [Perkins, et al., Antimicrob. Agents Chemother.42: 843-848 (1998)]. Although antiprotozoan activity appears not to be correlated with immunosuppressive or PPIase inhibitory activity [Bell, et al., Biochem. Pharmacol. 48:495-503 (1994); Khattab, et al., Exp. Parasitol. 90:103-109 (1998)], the protozoan cyclophilin, complexed to CsA or its non-immunosuppressive peptidic analogues, has been proposed to play an active role in mediating the antiparasitic effects of cyclophilin ligands [Berriman and Fairlamb, Biochem. J. 334:437-445 (1998)].
CyA and its non-immunosuppressive analogues also inhibit reproduction of filarial parasites in vivo with a potency unrelated to their immunosuppressive activity and their activity against Plasmodium [Zahner and Schultheiss, J. Helminthol. 61:282-90 (1987)], and have been shown to exert direct antihelmintic effects [McLauchlan, et al., Parasitology 121:661-70 (2000)].
CsA has also been found to be useful in affecting the viral replication process of the HIV-1 virus. The infectivity of the HIV-1 virus is believed to depend critically upon an interaction of the viral Gag polyprotein capsid complex with host Cyclophilin A. [Streblow et al. Virology 1998: 245, 197-202; Li et al. J. Med. Chem. 2000: 43,1770-9 ].
The aforementioned biological activities, which are believed to depend on the binding of a cyclophilin ligand to the native cyclophilin protein, may be of great therapeutic value in treating a range of medical conditions in animals, including humans. However, it is not desirable to treat conditions related to hair loss, mitochondrial energy breakdown, or HIV- or parasitic infections with an immunosuppressant cyclophilin ligand such as cyclosporin A. Furthermore, cyclophilin ligands known in the art to date are large molecules based on the peptidic structure of CsA. There thus exists an unmet need for non-immunosuppressive small molecule ligands of cyclophilin-type immunophilin proteins which are useful in the prevention or therapy of disease conditions relating to hair loss, breakdown of mitochondrial energy metabolism, HIV-infection, and infection with protozoan and helmintic parasites.
The present invention provides methods of preventing or retarding hair loss in patients undergoing therapy with doxorubicin, carboplatin, cisplatin, cyclophosphamide, dactinomycin, etoposide, hexamethamelamine, ifosfamide, taxol, vincristine, bleomycin, 5-fluorouracil, and other agents useful in the therapy of cancer, comprising administering to said patients an effective amount of a compound of formula I or II: 
where n in Cn is 0 or 1;
the dashed bond symbol represents an optional bond;
X and Y may independently be N, NH, O, S, or a direct bond;
R1 is the same or different from R2, and either can be
one or more C1-C6 branched or straight chain alkyl or alkenyl groups;
one or more C1-C3 branched or straight chain alkyl groups substituted by one or more Q groups;
or one or more Q groups,
where Q, which is optionally saturated, partially saturated, or aromatic, is a mono-, bi-, or tricyclic, carbo- or heterocyclic ring, wherein each ring may be optionally substituted in one to five positions with halo, hydroxyl, nitro, trifluoromethyl, acetyl,
aminocarbonyl, methylsulfonyl, oxo, cyano, carboxy, C1-C6 straight or branched chain alkyl or alkenyl, C1-C4 alkoxy, C1-C4 alkenyloxy, phenoxy, benzyloxy, amino, or a combination thereof, and wherein the individual ring sizes are 5-6 members, and wherein each heterocyclic ring contains 1-6 heteroatoms selected from the group consisting of O, N, S, or a combination thereof;
and R3 many be one to three substituents chosen from the group consisting of halo, hydroxyl, nitro, trifluoromethyl, C1-C6 straight or branched chain alkyl or alkenyl, C1-C4 alkoxy, C1-C4 alkenyloxy, phenoxy, benzyloxy, amino, Q as defined above, or a combination thereof; 
xe2x80x83where R4 and R5 may independently be
xe2x80x94Nxe2x80x94SO2xe2x80x94R,
xe2x80x94SO2xe2x80x94NRR,
xe2x80x94Oxe2x80x94R,
xe2x80x94COxe2x80x94Nxe2x80x94R,
xe2x80x94Nxe2x80x94COxe2x80x94R,
xe2x80x94COxe2x80x94R,
wherein each R may independently be hydrogen, Q, or a C1-C6 branched or straight alkyl or alkenyl chain, which may be substituted in one or more positions by C3-C8 cycloalkyl or cycloalkenyl, hydroxyl, or carbonyl oxygen, and where in said alkyl or alkenyl chain one or more carbon atoms are either optionally substituted with Q, or optionally replaced by O, S, SO, SO2, N, or NH; where Q, which is optionally saturated, partially saturated, or aromatic, is a mono-, bi-, or tricyclic, carbo- or heterocyclic ring, wherein each ring may be optionally substituted in one to five positions with halo, hydroxyl, nitro, trifluoromethyl, acetyl, aminocarbonyl, methylsulfonyl, oxo, cyano, carboxy, C1-C6 straight or branched chain alkyl or alkenyl, C1-C4 alkoxy, C1-C4 alkenyloxy, phenoxy, benzyloxy, amino, or a combination thereof, and wherein the individual ring sizes are 5-6 members, and wherein each heterocyclic ring contains 1-6 heteroatoms selected from the group consisting of O, N, S, or a combination thereof.
The present invention further provides a method of promoting hair growth in patients suffering from hair loss associated with treatment with one or a combination of the aforementioned chemotherapeutic agents, comprising administering to said patients an effective amount of a compound of Formula I or II.
The present invention further provides a method of preventing or retarding hair loss in patients undergoing radiation therapy, comprising administering to said patients an effective amount of a compound of Formula I or II.
The present invention further provides a method of promoting hair growth in patients suffering from hair loss associated with radiation therapy, comprising administering to said patients an effective amount of a compound of Formula I or II.
The present invention further provides a method of promoting hair growth in patients suffering from alopecia areata, androgenetic alopecia/male pattern baldness, anagen effluvium, trichotillomania, traction alopecia, telogen effluvium, and hair loss induced by drugs such as, for example, methotrexate, nonsteroidal anti-inflammatory drugs, or beta blockers, comprising administering to said patients an effective amount of a compound of Formula I or II.
For these purposes, the compounds may be administered as part of pharmaceutical or cosmetic compositions, singly, in combination with other compounds of the invention, in combination with other hair growth-promoting or hair-loss preventing agents, or in combination with one or several other active agents such as, for example, antibiotic agents, antidandruff agents, and anti-inflammatory agents. Thus, the invention provides pharmaceutical or cosmetic compositions especially formulated for topical application to the skin.
The invention further provides a method of blocking the mitochondrial permeability transition pore, comprising contacting the mitochondrion with a compound of Formula I or II.
The invention further provides a method of inhibiting breakdown of mitochondrial metabolism in cells which undergo oxidative stress, comprising contacting said cells with a compound of formula I or II.
The invention further provides a method of preventing or delaying cell death in a cell subjected to calcium overload, comprising contacting said cell with a compound of Formula I or II.
The invention further provides a method of preventing, mitigating, or delaying excitotoxic or hypoglycemic injury to cells, tissues or organs both in vitro and in vivo, comprising contacting said cells, tissues, or organs with a compound of Formula I or II.
The invention further provides a method of inhibiting breakdown of energy metabolism and cell death of mammalian cells following physiological induction of programmed cell death, comprising contacting said cells with a compound of Formula I or II.
The invention further provides a method of inhibiting breakdown of energy metabolism and cell death of mammalian cells following physiological stress related to hypoxia, hypoglycemia, excitotoxic insult, or calcium overload, comprising contacting said cells with a compound of Formula I or II.
The invention further provides a method of preventing or delaying the death of cells in large scale/commercial scale cell culture, comprising contacting said cells with a compound of Formula I or II.
The invention further provides a method of using a compound of Formula I or II in the diagnosis, cure, mitigation, treatment, or prevention of ischemic injury or ischemia/reperfusion injury, comprising administering to a patient at risk for, or suffering from, an ischemic or ischemia/reperfusion injury an effective amount of a compound of Formula I or II.
The invention further provides a method of using a compound of Formula I or II in the diagnosis, cure, mitigation, treatment, or prevention of ischemic injury or ischemia/reperfusion injury, comprising administering to a patient at risk for, or suffering from, an ischemic or ischemia/reperfusion injury an effective amount of a compound of Formula I or II, wherein the ischemic injury or ischemia/reperfusion injury is selected from the group consisting of mesenteric infarction, bowel ischemia, hepatic infarction, renal infarction, splenic infarction, and ischemic heart disease.
The invention further provides a method of using a compound of Formula I or II in the diagnosis, cure, mitigation, treatment, or prevention of ischemic injury or ischemia/reperfusion injury, comprising administering to a patient at risk for, or suffering from, an ischemic or ischemia/reperfusion injury an effective amount of a compound of Formula I or II, wherein the ischemic injury or ischemia/reperfusion injury is related to congestive heart failure, myocardial ischemia, or coronary heart disease.
The invention further provides a method of treating an ophthalmic disorder in an animal, comprising administering to said animal a therapeutically effective amount of a compound of Formula I or II.
The invention further provides a method of treating an ophthalmic disorder in an animal, comprising administering to said animal a therapeutically effective amount of a compound of Formula I or II, wherein said ophthalmic disorder is glaucoma, ischemic retinopathy, vascular retinopathy, or degeneration of the photoreceptor cell layer.
The invention further provides a method of treating Reye""s syndrome in a patient, comprising administering to said patient a therapeutically effective amount of a compound of Formula I or II.
The invention further provides a method of preventing or reducing tissue damage of organs used in organ transplantation surgery, comprising contacting said organs with a compound of Formula I or II.
The invention further provides a method of treating an infection with pathogenic protozoan or helmintic parasites, comprising contacting said parasites with a compound of Formula I or II.
The invention further provides a method of treating an infection with pathogenic protozoan or helmintic parasites in an animal, comprising administering to said animal a therapeutically effective amount of a compound of Formula I or II.
The invention further provides a method of treating a medical condition related to infection with pathogenic protozoan or helmintic parasites in an animal, comprising administering to said animal a therapeutically effective amount of a compound of Formula I or II, wherein said medical condition is malaria, river blindness, lymphatic filariasis, intestinal roundworm infection, tapeworm infection, pinworm infection, toxoplasmosis, leishmaniasis, trypanosomiasis, or bilharzia.
The invention further provides a method for treating infection with a virus of the HIV type in a patient, comprising administering to said patient a therapeutically effective amount of a compound of Formula I or II.
The invention further provides a method for treating acquired immune deficiency syndrome (AIDS) in a patient, comprising administering to said patient a therapeutically effective amount of a compound of Formula I or II.
A number of compounds can be selected for use from Formulae I and II. For example, starting with a particular compound, any of the individual variable groups R1-R5, X, Y, and a value for xe2x80x98nxe2x80x99 can be selected while one or more of the other variable groups can be modified. For example, in Formula I, the xe2x80x9cnxe2x80x9d can be set at 0 to select subgroups of related compounds with X and Y being both NH, or both being O, or X being NH and Y being O, and within each of those 3 groups R3 being present or absent, and then within each of those 6 groups the 6-membered ring structure is either a cyclohexyl or an aromatic ring, which results in 12 subgroups of related compounds. Any of those 12 subgroups can be selected and further divided into additional subgroups of compounds defined by having an R1 the same as R2 or by having both R1 and R2 comprise a substituted benzyl or substituted phenyl group. This process can be repeated using any one or combination of the variable groups. In this way, one skilled in the art can select and use groups of related compounds or even individual compounds, all within the invention. Many examples are shown below; however, they are merely representative of the scope of changes and modifications possible. One skilled in the art can devise many separate compounds from the description of the Formulae alone. Thus, the invention specifically includes numerous individual compounds that fall within the definition of either Formula I or II.
Compounds of Formulae I and II may be prepared or formulated as a salt or derivative for some uses, including pharmaceutical and tissue or cell culture uses. The compounds of the invention can also be part of a composition comprising one or more compounds of Formula I or II. Thus, pharmaceutically acceptable salts and derivatives of any of the compounds, or compositions comprising them, are specifically included in this invention. A compound of Formula I or II, or a compound having Formulae I or II, will optionally include the salt or derivative of the compound depicted in the formula.
The compounds of the invention can be produced as a mixture of isomers or racemic mixtures or as optically pure compounds. Methods for separating stereoisomers can also be used to enrich mixtures for one or more compounds. The compositions of the invention may similarly contain mixtures of stereoisomers, mixtures of one or more stereoisomers, or be enriched for one or more stereoisomers. All of these forms are specifically included in this invention.
Preferably, compounds of Formulae I and II selectively bind to a CyP as detected, for example, by a measurable inhibition of the rotamase (PPIase or peptidyl-prolyl cis-trans isomerase enzyme) activity of CyP. xe2x80x9cSelectively bind to a CyPxe2x80x9d means the compounds do not possess a significant binding affinity toward a FKBP and/or do not possess a biological activity associated with binding to a FKBP. For example, the IC50 towards FKBP is at or above 10 xcexcM or at or above 50 xcexcM. The skilled artisan is familiar with ways to detect rotamase inhibition in CyP and FKBP. In addition, a number of ways for detecting binding to a CyP are described below.
As is readily apparent from Formulae I and II, a common 1-,3-substitution pattern on a central ring structure exists. This common pattern differs from the approaches previously taken to identify other immunophilin binding compounds or drugs. For example, Holt et al. (Bioorg. Med. Chem. Letters, 4: 315-320 (1994)) discuss a pipecolate, or 1-(1,2-dioxo) 2-carboxylate piperidine containing base structure for binding to FKBP. Similarly, earlier work by the inventors established the relevance of a 1-(1,2-dioxo) 2-carboxylate pyrrolidine containing structure for binding to FKBP (Steiner et al., PNAS 94:2019-2024 (1997)). Presumably, these structures mimic the natural substrate for the rotamase activity, a proline-containing fragment of a protein. In a protein, the amino acid proline corresponds to a 1,2-substituted pyrrolidine structure. Prior work has generally incorporated that structure. However, Formulae I and II do not correspond to a 1,2-substituted pyrrolidine structure. Yet, as demonstrated here, compounds of these formulae possess important bioactive and biochemical functions.
The body of work related to analogues of cyclosporin A, FK-506, and rapamycin further distances the compounds of this invention from prior work. (See, for example, U.S. Pat. Nos. 5,767,069, 5,284,826, 4,703,033, and 5,122,511.) These analogues typically possess a cyclic peptide structure.
In another aspect, the invention relates to methods for binding non-peptidic compounds to cyclophilin-type immunophilins. Binding results in an xe2x80x9cimmunophilin:drugxe2x80x9d complex, which is considered to be the active agent in the in vivo immunosuppressive and neurotrophic activities of rotamase inhibitors (Hamilton and Steiner, J. of Med. Chem. 41:5119-5143 (1998); Gold, Mol. Neurobiol. 15:285-306 (1997)). Whether or not the complex acts for any or all the therapeutic actions of these rotamase inhibitors, focusing on the immunophilin:drug interaction has led to the discovery a number of new drug compounds. Accordingly, methods of using compounds, such as those of Formulae I and II, to create an immunophilin:compound complex, or a CyP:compound complex, provides an important aspect of this invention. This aspect can be exploited, for example, in methods where the compound, or a mixture comprising one or more of the compounds of the invention, is administered to cells in culture or to an animal.
While the immunophilin:compound complex has beneficial effects in vivo and in cultured cells, numerous other uses for binding the compounds to an immunophilin exist. For example, in vitro binding experiments can be used to identify and purify cellular components that interact with the immunophilin complex. An affinity chromatography column or matrix bearing the compound can be reacted with a CyP, and cellular or tissue extracts passed over the column or matrix.
Thus, the invention also provides methods for forming immunophilin:compound or CyP:compound complexes as well as the complexes themselves. To form these complexes, the compounds can contact an immunophilin or CyP protein in vivo, in vitro, or within a cell. In preferred embodiments, the compound contacts a human CyP protein, such as one or more of CyP A, B, C, or D. The CyP protein can be native to the cell or organism, produced via recombinant DNA, produced by other manipulations involving introduced genetic material, or produced by synthetic means. Furthermore, chimeric proteins possessing immunophilin domains that function to bind immunophilin ligands can also be used to form a protein:compound complex. The formation of the CyP:compound, immunophilin:compound, or protein:compound complex need not be irreversible.
The binding of a compound to a CyP can be detected in a number of ways, including rotamase inhibition assay, affinity chromatography, in vivo cardioprotection assay, in vitro mitochondrial permeability transition assay, or by any of the activities in experimental models of hair loss, parasitic infection, stroke, ischemia and reperfusion injury as described below, in the examples, or in the cited references.
The invention also provides compositions comprising at least one compound of Formula I or II. The compositions may comprise one or more pharmaceutically acceptable carriers, excipients, or diluents. These compositions, or the compounds themselves or mixtures of them, can be administered to an animal. Administration can be one method to allow the compound to contact a CyP within the animal. As one skilled in the art would recognize, various routes of administration are possible. Exemplary routes are specifically described in the detailed description below.
The following detailed description should not be taken as a limitation on the scope of the invention. The embodiments and examples given are illustrative of the invention. Additional aspects of the invention can be devised by reference to this disclosure as a whole in combination with the references cited and listed throughout and at the end of the specification and the knowledge of one skilled in the art. All of the references cited and listed can be relied on, in their entirety, to allow one to make and use these additional aspects of the invention.